Selective targeting of cancer tissues can be achieved by tumour-tropic organisms, including certain replication competent viral vectors and bacteria. Such organisms are generally antineoplastic in their own right, and a number are in clinical trials (or clinical use) as novel therapeutic agents. Ideally such agents would be introduced via systemic administration, and would “seek out” cancerous tissues. However, applications to date have been limited owing to an inability to non-invasively image the location of viruses or bacteria in the body post-administration. The self-amplifying nature and uncertain tropism for human tissues has hampered the selection and development of oncolytic viruses and bacteria.
Non-Invasive Imaging Methods for Biological Vectors
Tissue biopsies and other invasive approaches to imaging tumour-tropic biological vectors cannot be applied to all organs of the body in concert and repeated sampling is rarely clinically feasible. However, the requirement for repeat sample analysis is necessary for dynamic agents that amplify and can redistribute micro-regionally and systemically with time, and mandates a non-invasive methodology that can be applied at regular intervals. This is desirable to allow early intravenous administration of novel vectors in human clinical trials. Of note, animal toxicological models are generally considered to have poor predictive value for human tropic viruses and consequently there is a need to monitor experimental vectors thereby establishing early proof of principle in (preclinical) animal models and in human trials.
Various indirect reporter gene approaches have been tried in an attempt to monitor vector behaviour in living systems including bioluminescence, fluorescence and secreted plasma markers, none of which are considered clinically viable for various reasons including signal attenuation or lack of spatial information.
Positron Emission Tomography (PET) technology is increasingly being applied to the area of therapy development and is the most attractive method for non-invasive and comprehensive measurement of whole body vector distribution. Multiple sampling from the same patient is also possible. PET is safe, accurate and results are reproducible. It also has extremely high sensitivity to imaging probe molecules and is ideal for monitoring cellular or molecular events early in the course of the disease, during therapy, and for evaluating disease recurrence.
PET-based vector imaging has been achieved in preclinical studies for the reporter gene Herpes simplex virus thymidine kinase (HSV-tk) Bennett et al, 2001, Nat Med 7 (7): 859-863; Gambhir et al, 2000, Proc Natl Acad Sci USA 97 (6): 2785-2790; Soghomonyan et al, 2005, Cancer Gene Ther 12 (1): 101-108) and proof of principle studies are underway with newly designed HSV-tk PET probes (Hackman et al, 2002, Molec Imag 1 (1): 36-42; Jacobs et al, 2001, Cancer Res 61 (7): 2983-2995; Min et al, 2003, Eur J Nuc Med Mol Imaging 30 (11): 1547-1560; Miyagawa et al, 2008, J Nucl Med 49 (4): 637-648) including FHBG (9-(4-[18F]fluoro-3 hydroxymethylbutyl)guanine). However, it has been demonstrated that tumour retention of 18F-FHBG, monitored via PET, was unsuccessful in predicting HSV-1tk virus load due to tumour release of soluble phosphorylated 18F-FHBG following tumour cell oncolysis (Kuruppu et al, 2007, Cancer Res 67 (7): 3295-3300). In addition, imaging is hampered using current probes by excessive background signal and a lack of homogenous distribution throughout the body. Other disadvantages to known systems include laborious synthesis of the probes, that the probes can themselves be toxic, and easy degradation of probe molecules in the blood, limiting the ability for systemic administration.
Use of Bacterial Nitroreductases as Reporter Genes for Imaging
Bacterial nitroreductases (NTRs) can catalyse the reduction of certain nitroheterocyclic/nitrocarbocyclic/nitroaromatic molecules. Limited studies have been conducted on their utility as enzymes for reporter gene systems. Available publications and patents relating to imaging are restricted to the use of fluorescent probe substrates with minimal clinical utility. For example, the non-fluorescent compound 6-chloro-9-nitro-5H-benzo[a]phenoxazin-5-one (C-22220, CNOB) has been described as a fluorogenic probe for detection of nitroreductase activity (Molecular Probes Handbook, Ed. Richard P. Haugland, 10th Edition, 2005, p 535). Escherichia coli NfsB can metabolise CNOB to a fluorescent aminophenoxazine (Ex/Em 617/625 nm) and CNOB has been used for the detection of E. coli nfsB expression in tumour bearing nude mice injected with E. coli NfsB-expressing Clostridia sporogenes spores (Liu et al, 2008, Cancer Res 68 (19): 7995-8003). However, E. coli NfsB has limited catalytic flexibility and NfsB has previously been found to be inactive when evaluated with 2-nitroimidazole (2-NI) substrates (Anlezark et al, 1995, Biochem Pharmacol 50 (5): 609-618). The scarcity of characterised microbial NTR genes and their coordination with appropriate prodrug substrates is an unaddressed limitation.
The non-fluorescent 6-nitroquinoline has been described as a fluorogenic probe for the detection of E. coli nfsB expression in cell culture monolayers (Singleton et al, 2007, Cancer Gene Ther 14 (12): 953-967). In a further example, CytoCy5 is a cell-entrapped red fluorescent probe for E. coli NfsB with recently demonstrated utility in cell lines and animal models (U.S. Pat. No. 7,579,140 Bhaumik et al, 2011, Gene Ther July 14; epub ahead of print). However, despite recent research on these systems, they are still deemed to be inadequate as nitroreductase-based reporter gene systems for clinical applications due to problems including signal attenuation and lack of spatial information.
Thus it is desirable to provide alternative non-invasive imaging technologies that preferably allow for rapid, reproducible and quantitative imaging and/or that enable the monitoring of gene/vector distribution and amplitude in the same patient or animal over time. Additionally, there would be an advantage in providing imaging technologies to monitor the spatial and temporal distribution of vector systems with time in a manner that is predictive of normal tissue toxicity and antitumour efficacy.
Gene-Directed Enzyme Prodrug Therapy (GDEPT)
Gene-directed enzyme prodrug therapy (GDEPT) is a gene therapy strategy in which a therapeutic gene encodes an exogenous enzyme that will convert an administered non-toxic prodrug into an active cytotoxic derivative. GDEPT is made up of three components; the prodrug to be activated, the prodrug activating enzyme, and the delivery vector for the corresponding gene. Preferential activation of the prodrug in transduced tumour cells generates high intra-tumoural drug (activated prodrug metabolite) concentrations and therefore increases the therapeutic index of the drug.
It would be preferable to be able to utilise a single enzyme or gene product to enable both imaging and prodrug activation as imaging may directly predict the location and magnitude of prodrug activation, providing critical safety information prior to introduction of a conditionally cytotoxic therapy component.
Selectivity for tumour (over normal) tissues is predicated on the use of a biological vector, such as an oncolytic virus, that has been targeted to the tumour tissues. Therapy that utilises viral delivery vehicles is also known as virus-directed enzyme prodrug therapy (VDEPT). Alternatively, use of bacterial vectors tropic for tumour tissues, such as Clostridia sp., Salmonella sp. or Bifidobacter sp. is commonly termed bacterial-directed enzyme prodrug therapy (BDEPT), or in certain specific cases CDEPT (for Clostridia-directed enzyme prodrug therapy). These are all vector specific variants of GDEPT and are considered to be covered by this common acronym. An additional term, ADEPT, refers to antibody-directed enzyme prodrug therapy and encompasses the use of epitope-specific antibodies to guide systemically administered antibody-enzyme fusions to tumour sites in order to target prodrug activation.
The limited activity of GDEPT systems has led to the evaluation of the E. coli nitroreductase NfsB in combination with CB1954 (5-aziridinyl-2,4-dinitrobenzamide) and various other nitroheterocyclic/nitrocarbocyclic/nitroaromatic prodrugs (Denny, 2002, Curr Pharm Des 8 (15):1349-1361; Searle et al, 2004, Clin Exp Pharmacol Physiol 31 811-816; Singleton et al, 2007, Cancer Gene Ther 14 (12): 953-967). The NfsB/CB1954 combination has undergone evaluation in a VDEPT setting with some signs of activity (Palmer et al, 2004, J Clin Oncol 22 (9): 1546-1552). Alternate NTRs, an evolved form of E. coli YieF (Barak et al, Mol Can Ther 5 (1): 97-103) and wild-type E. coli NfsA (Vass et al, 2009, Br J Cancer 100 (12): 1903-1911; Prosser et al, 2010, Biochem Pharmacol 79, 678-687) have been evaluated in combination with CB1954 (and the former also with mitomycin C and CNOB (C-22220) (Thorne et al, 2009, Mol Can Ther 8 (2): 333-341)). Bacillus amyloliquefaciens YwrO and Enterobacter cloacae NR are also known to reduce the prodrug CB1954 (Anlezark et al, 2002, Microbiology 148 (Pt 1): 297-306).
The currently known and studied bacterial nitroreductase enzymes for GDEPT have not been shown to be capable of metabolising 2-nitroimidazole PET imaging agents, rendering them ineffectual as reporter genes for non-invasive imaging of gene/vector distribution and amplitude in the same patient or animal over time. Additionally, CB1954 has low potency, poor formulation characteristics, an insufficient bystander effect for meaningful therapeutic utility and is poorly tolerated in humans. E. coli NfsB possesses poor enzyme kinetic properties with respect to CB1954 reduction and has limited substrate flexibility. Attempts to monitor NfsB activity in murine tumour xenografts using CNOB (C-22220) have required direct intratumour injection of fluorogenic substrate. Use of nfsB-labelled virus in humans has necessitated direct intratumoural injection since monitoring of nfsB/virus distribution following systemic administration is not possible.
The ability to ablate cells without localised damage to neighbouring tissue (known as single cell ablation) is seen as a valuable safety control for enabling the elimination of a vector in the matrix, cells or tissues should this be deemed necessary. The ability to control viral (VDEPT) or bacterial (BDEPT) infection is an additional biosafety feature and is considered to be a desired design feature in replicating biological vectors. To achieve this, activation of prodrugs that provide reduced, substantially minimal or zero bystander effect is also desirable.
Thus there is a need for nitroreductases that are more catalytically efficient and which can utilise a broad array of prodrugs that are able to distribute well in tumour tissues. Further there is a need for nitroreductases that can be imaged prior to prodrug administration to determine tissue distribution since the combination of nfsB/virus and prodrug is specifically designed to be cytotoxic upon interaction.
It is an object of the invention to overcome or ameliorate at least one of the disadvantages of the prior art, or at least to provide the public with a useful choice.